Recent trends favoring organic and sustainable turfgrass management practices have led to an increased desire for biologically based alternatives to traditional synthetic herbicides. Thaxtomin A, produced by the bacterium Streptomyces scabies, has been reported to have PRE efficacy on broadleaf weeds, but efficacy of thaxtomin A on annual grassy weeds and safety to newly seeded cool-season turfgrasses have not been reported. Field experiments were conducted to evaluate PRE efficacy of thaxtomin A on smooth crabgrass and annual bluegrass. Monthly applications of thaxtomin A from April to July controlled smooth crabgrass through July but did not provide season-long control equivalent to an industry standard PRE herbicide. An initial application of thaxtomin A at 380 g ai ha−1 followed by two applications at 190 or 380 g ha−1 at 4-wk intervals provided season-long annual bluegrass control similar to an industry standard PRE herbicide. At 380 g ha−1, thaxtomin A reduced tall fescue and perennial ryegrass cover when applied 1 wk before seeding, at seeding, or 1 wk after seeding but was safe at other application timings. Up to three applications of thaxtomin A at 380 g ha−1 at 4-wk intervals did not reduce perennial ryegrass cover. Applications to creeping bentgrass resulted in unacceptable turfgrass injury. These results suggest that thaxtomin A can suppress annual grassy weeds in tall fescue or perennial ryegrass turf when applied at least 2 wk before or after seeding. Furthermore, repeated applications of thaxtomin A can provide effective PRE control of annual bluegrass during overseeded perennial ryegrass establishment.